Vasoactive Intestinal Polypeptide Type-1 Receptor Regulation DESENSITIZATION, PHOSPHORYLATION, AND SEQUESTRATION*

The vasoactive intestinal polypeptide type-1 (VPAC 1 ) receptor is a class II G protein-coupled receptor, distinct from the adrenergic receptor superfamily. The mechanisms involved in the regulation of the VPAC 1 receptor are largely unknown. We examined agonist-dependent VPAC 1 receptor signaling, phosphorylation, desensitiza- tion, and sequestration in human embryonic kidney 293 cells. Agonist stimulation of cells overexpressing this receptor led to a dose-dependent increase in cAMP that peaked within 5–10 min and was completely desensitized after 20 min. Cells cotransfected with the VPAC 1 receptor (VPAC 1 R) and G protein-coupled receptor kinases (GRKs) 2, 3, 5, and 6 exhibited enhanced desensitization that was not evident with GRK 4. Immunoprecipitation of the epitope-tagged VPAC 1 receptor re- vealed dose-dependent phosphorylation that was increased with cotransfection of any GRK. Agonist-stim-ulated internalization of the VPAC 1 R peaked in 10 min, and neither overexpressed (cid:1) -arrestin nor its dominant-negative mutant altered internalization. However, a dynamin-dominant negative mutant did inhibit VPAC 1 receptor internalization. Interestingly, VPAC 1 R specificity in desensitization was not evident by study of the overexpressed receptor; however, we determined that human embryonic kidney 293 cells express an endogenous VPAC 1 R that did demonstrate dose-dependent a similar pattern (data not shown). To investigate the role of (cid:1) -arrestin in VPAC 1 receptor internalization, we used immunofluorescence to study the fate of the VPAC 1 receptor and (cid:1) -arrestin after agonist stimulation.

The neuromodulator vasoactive intestinal polypeptide (VIP) 1 is a potent vasodilator and has been shown to participate in regulating gastrointestinal motility, enzyme secretion, and blood flow (1)(2)(3). The type-1 VIP (VPAC 1 ) receptor is a member of a family of G protein-coupled receptors (GPCRs), designated as class II. These receptors share a significant degree of sequence homology within the family (Ͼ50%), but are distinct from members of the larger rhodopsin/adrenergic receptor family (class I) (4). GPCRs are membrane proteins characterized by seven transmembrane-spanning domains and are named for their functional interaction with heterotrimeric guanine nucleotide-binding regulatory proteins (G proteins). Agonist-activated GPCRs transduce extracellular signals into intracellular events through activation of G protein-regulated second messenger pathways or ion channels. Agonist activation of GPCRs also leads to the competing process whereby uncoupling of the receptor from its G protein results in attenuation, or desensitization, of signaling events (5). An important process in the desensitization of GPCRs is the phosphorylation of agonistoccupied receptors, followed by receptor internalization and, finally, eventual recycling to the plasma membrane as competent receptors (5).
G protein-coupled receptor kinases (GRKs) contribute to desensitization of GPCRs by phosphorylating agonist-activated receptors (6). Second messenger-dependent protein kinases, such as cAMP-dependent protein kinase and protein kinase C, can also phosphorylate GPCRs and dampen signaling; however, these processes are independent of receptor occupancy. GRK-mediated receptor phosphorylation promotes subsequent binding of arrestin proteins. Arrestins are cytosolic proteins that bind GRK-phosphorylated receptors to prevent G protein coupling, thereby quenching intracellular signaling, and that target GPCRs to clathrin-coated pits for internalization/sequestration, dephosphorylation and recycling (7). The mechanisms regulating these various processes are critical to the normal function of GPCRs.
We have previously demonstrated that the secretin receptor, also a class II GPCR, is regulated differently from many class I receptors (8,9). Like the ␤ 2 AR (class I), phosphorylation and desensitization of the secretin receptor was promoted by GRKs (9); however, unlike the ␤ 2 AR, sequestration of secretin receptors was not increased by GRK overexpression nor was sequestration inhibited by a dominant negative (V53D) ␤-arrestin mutant (8). Instead, second messenger-dependent phosphorylation was important for sequestration of the secretin receptor, whereas GRKs and ␤-arrestin are critical to the internalization of the ␤ 2 AR (class I) (8,10).
In this paper we probe the roles of receptor phosphorylation by GRKs and ␤-arrestin recruitment in the regulation of VPAC 1 receptor signaling, desensitization, and sequestration. Moreover, we determined that human embryonic kidney (HEK 293) cells express an endogenous VPAC 1 receptor and used this receptor to demonstrate GRK specificity that was not evident * This work was supported in part by National Institutes of Health Grants 5T32DK07568 and DK02544 and by the Mal Tyor Scholarship Award in Gastroenterology (to M. A. S.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18  in overexpressed systems. These findings suggest that the VPAC 1 receptor is regulated by agonist-stimulated, GRK-mediated receptor phosphorylation, ␤-arrestin translocation, and dynamin-dependent receptor internalization likely via clathrin-coated pits.

EXPERIMENTAL PROCEDURES
Materials-General chemicals and reagents were from Sigma. Vasoactive intestinal polypeptide was obtained from Peninsula Laboratories. HEK 293 cells were obtained from the American Tissue Culture Collection. Tissue culture supplies were obtained from Invitrogen. Labeled vasoactive intestinal polypeptide ( 125 I) was prepared and purified by high performance liquid chromatography (9,11). [2,  Plasmid Preparation-The full-length nucleotide sequence of the rat VIP (VPAC 1 ) receptor (12) was amplified from rat heart cDNA by PCR using gene-specific oligonucleotides. An epitope-tagged rat VPAC 1 R was prepared as described for the rat secretin receptor (9). The FLAG epitope was placed on the N-terminal region of the mature receptor following a modified influenza hemagglutinin signal sequence to produce a protein that could be recognized with commercially available anti-FLAG antibodies. The cDNAs were inserted into the pcDNA 1/Amp plasmid (Invitrogen) using HindIII and XbaI. Fidelity was demonstrated by dideoxy sequencing. GRK cDNAs were produced as previously described: GRKs 2 and 3 (13), GRK 4 (14), GRK 5 (15), and GRK 6 (16). ␤-Arrestin-1 and dynamin, as well as their dominant negative mutants, were used as previously described (8). ␤-Arrestin-green fluorescent protein (GFP) was produced as outlined by Barak et al. (17). Plasmid purification was performed with Qiagen reagents.
Transfection-Transient transfections were performed with calcium phosphate co-precipitation. One to 10 g of vector DNA was transferred into a 6-ml Falcon tube with 450 l of sterile water and 50 l of 2.5 M CaCl 2 . Then 500 l of 2ϫ HEPES-buffered saline (0.28 M NaCl, 0.05 M HEPES, 1.5 mM Na 3 PO 4 , pH 7.1) was added to the tube and mixed well. This mixture was added dropwise to the 100-mm dish of cells.
Membrane Preparation/Binding-All steps were performed at 4°C. Plates were placed on ice, media aspirated, and cells washed with 10 ml of ice-cold PBS. Five to 10 ml of lysis buffer (10 mM Tris, 5 mM EDTA with protease inhibitors: 10 g/ml aprotinin, 5 g/ml leupeptin, 0.7 g/ml pepstatin A, 10 g/ml benzamidine, 0.2 mM phenylmethylsulfonyl fluoride) were added to each plate. With a cell lifter, cells were scraped off the plate and placed in 15-ml conical tubes on ice. Cell fragments were homogenized with a Polytron PT 3000 for 20 -30 s at 14,000 -16,000 cps. Material was centrifuged at 300 -400 ϫ g for 10 min to remove unlysed cells and nuclei. Supernatant was transferred to 13 ϫ 100-mm tubes on ice and centrifuged at 18,000 rpm (40,000 ϫ g) (Sorvall SM24 rotor) for 30 min at 4°C. Supernatant was discarded, and the membrane pellet was resuspended in binding buffer, for immediate assay, or lysis buffer and stored at Ϫ80°C.
Membrane binding was performed as published (11). Briefly, using constant amount of HEK 293 membrane protein, competition displacement (using synthetic vasoactive intestinal polypeptide, Peninsula Laboratories) of 125 I-VIP binding was performed in triplicate tubes. Nonspecific binding was defined in the presence of 1 M unlabeled VIP. Data were analyzed using Graph Pad-Prism and LIGAND software as described (9,11).
Adenylyl Cyclase Assays-The accumulation of cAMP in intact cells was quantified chromatographically by the method of Salomon (18). Cells transiently transfected with the VPAC 1 receptor or untransfected were plated to a density of ϳ2-3 ϫ 10 5 cells/well and labeled with [ 3 H]adenine (1 Ci/ml) in MEM, 5% FBS, 50 mg/liter gentamicin (1 ml/well) for 12-16 h prior to experimentation. Labeling medium was aspirated, and cells were washed with 1 ml of PBS and preincubated in assay medium (1 ml/well, MEM, 0% FBS, 10 mM HEPES, 1 mM isobutylmethylxanthine) for 15-30 min. Cells were stimulated with appropriate agonist, and, at the end of the experimental duration, medium was aspirated and 1 ml of ice-cold stop solution (0.1 mM cAMP, 4 nCi/ml [ 14 C]cAMP, 2.5% perchloric acid) was added to each well. Plates remained at 4°C for 20 -30 min, after which solution was transferred to 12 ϫ 75-mm tubes containing 100 l of 4.2 M KOH. Tubes were vortexed and stored at 4°C for cAMP determination by column chromatography (18). Data are normalized for total cellular uptake of [ 3 H]adenine and using [ 14 C]cAMP for column efficiency as previously described (18).
VIP Receptor Internalization by Immunofluorescence-HEK 293 cells transiently transfected with 5 g of cDNA for FLAG-tagged VPAC 1 R were plated onto 35-mm dishes containing a central glass well as described (17). Cells were maintained at 4°C to prevent receptor internalization while incubating with agonist (100 nM VIP), primary antibody (IgG-M2-FLAG, Eastman Kodak Co.) and secondary antibody (Fab conjugated with fluorescein isothiocyanate, Organon Teknika). Sequential incubations were 30 min in duration and occurred in the sequence listed. Cells were washed three times with cold PBS after each antibody application. Immediately following the last PBS wash, cells were viewed using confocal microscopy (basal time point), whereas a second plate of identically treated cells was warmed at 37°C for 1 h prior to imaging (60 min of treatment).
Immunofluorescent Colocalization of VIP Receptor with ␤-Arrestin-GFP-HEK 293 cells transiently transfected with 5 g of cDNA for wild-type VIP receptor were plated onto glass coverslips contained in six-well plates. After an initial wash, cells were stimulated with 100 nM VIP in a 37°C incubator. After 30 and 60 min, cells were fixed in 4% paraformaldehyde for 25 min. Cells were successively incubated for 1 h at room temperature with primary antibody (IgG-M2-FLAG) and secondary antibody (Texas Red-conjugated goat anti-mouse, Molecular Probes Inc.) dissolved in solubilization buffer consisting of 0.2% Triton X-100 and 1% bovine serum albumin in phosphate-buffered saline. Cells were washed for 20 min in solubilization buffer after each antibody incubation. Immediately following the last wash, glass coverslips containing the cells were mounted onto glass slides for viewing using a Zeiss laser scanning confocal microscope. ␤-Arrestin-GFP (1 g cDNA) was coexpressed with wild-type VPAC 1 (5 g of cDNA), and an identical protocol was followed except that cells were stimulated with VIP alone for only 2 min.
Immunoprecipitation of the VIP Receptor with ␤-Arrestin-HEK 293 cells transiently transfected with 3 g of cDNA for FLAG-tagged wildtype VIP receptor and 2 g of cDNA for ␤-arrestin were treated with medium alone or stimulated with 100 nM VIP in a 37°C incubator for 5, 10, or 20 min. One plate of HEK 293 cells was transfected only with 2 g of cDNA ␤-arrestin and served as the negative control. After stimulation, cells were lysed with lysis buffer (50 mM Tris, pH 8.0, 5 mM EDTA, 0.05% SDS, 200 mM NaCl, 10 mM NaF, 10 mM disodium pyrophosphate, and 1% Triton-X-100). Supernatant was immunoprecipitated with IgG-M2-FLAG antibody and separated on a 10% SDS-PAGE gel. After separation, proteins were transferred to nitrocellulose and immunoblotted with antibody to ␤-arrestin (31).
Western Blotting-Cellular proteins were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Protein was transferred to nitrocellulose and then subjected to immunoblotting with appropriate GRK antiserum (14,19,20). Blots were revealed by chemiluminescence.
Receptor Expression-In plasmid co-transfection experiments, receptor expression was determined by flow cytometry analysis of a sample from each transfection group (9). The fluorescence was determined by incubation for 1 h at 4°C with monoclonal IgG-M2-FLAG (1:600 dilution, Kodak), washed three times with PBS, and detected with Fc specific, fluorescein-labeled goat anti-mouse (1:200 dilution, Sigma). Cells were washed, removed from the plate with 10 mM Tris, pH 7.4, 5 mM EDTA and fixed with 3.6% formaldehyde. Samples were analyzed on a Becton-Dickson flow cytometer. Baseline fluorescence was determined from a sample of HEK 293 cells untransfected and/or a sample of HEK 293 cells transfected with the VPAC 1 not exposed to primary antibody (IgG-M2-FLAG). Baseline fluorescence was subtracted from each sample.
Receptor Internalization/Sequestration-Sequestration is defined as the number of receptors removed from the cell surface after agonist exposure, as determined by flow cytometry (8). Cells were plated to a density of ϳ1-1.5 ϫ 10 6 cells per well and exposed to agonist for the appropriate time. After washing with iced PBS, cells on ice were exposed for 1 h to monoclonal IgG-M2-FLAG antibody (1:600 dilution, Kodak) or 12CA5 (1:500 dilution, Roche Molecular Biochemicals), washed three times with PBS, and detected with Fc specific, fluoresceinlabeled goat anti-mouse antibody (1:200 dilution, Sigma). Cells were washed, removed from the plate with 10 mM Tris, pH 7.4, 5 mM EDTA and fixed with 3.6% formaldehyde. Samples were analyzed on a Becton-Dickson flow cytometer. Baseline fluorescence was determined from a sample of HEK 293 cells transfected with the VPAC 1 not exposed to agonist and another sample not exposed to primary antibody (IgG-M2-FLAG or 12CA5). Baseline fluorescence was subtracted from each sample.
Using RT-PCR to Identify the VIP Receptor Endogenously Expressed in HEK 293 Cells-RNA was prepared using RNAzol (Tel-Test, Inc., Friendswood, TX) and RT-PCR was performed using a PerkinElmer Life Sciences MMLV kit following the manufacturers' protocols. Primers were designed to distinguish between the two subtypes of VIP receptors as follows: human VPAC 1 receptor 463-486 (5Ј-GCCACCCT-TCTGGTCGCCACAGCT) and 1115-1092 (5Ј-TTCACTTCAGGCTTAA-AATTGTCC), and human VPAC 2 receptor 414 -438 (5Ј-ATGTCTCTT-GCAACAGGAAGCATA) and 1067-1044 (5Ј-TGGTATTTGGAGGAGA-TGCTGATG). PCR was initiated by adding 0.5 l of Taq polymerase (PerkinElmer Life Sciences). PCR was run at 96°C for 2 min, followed by 35 cycles of 30 s at 94°C, 30 s at 55°C, and 45 s at 72°C. Program was terminated with 7 min at 72°C. Samples were resolved with a 1% agarose gel and ethidium bromide and examined under UV light.
␤-Arrestin-GFP Translocation-Transfected HEK 293 cells were plated onto 35-mm dishes containing a central glass well as described (17). Cells in Dulbecco's modified medium, pH 7.4, buffered with 20 mM HEPES were stimulated with 1 M agonist while being viewed on a Zeiss laser scanning confocal microscope.
Receptor Phosphorylation-Cells were plated to a density of ϳ1-1.5 ϫ 10 6 cells/well and labeled with [ 32 P] orthophosphate (66 Ci/well) for 1 h in phosphate-free MEM, 20 mM HEPES, pH 7.4, at 37°C. Agonist was applied as indicated in figure legends. Treatment was stopped by placing the cells at 4°C and washing with ice-cold phosphate-buffered saline (3 ml/well) twice and then adding 400 l/well radioimmune precipitation buffer (150 mM NaCl, 50 mM Tris-HCl, pH 8, 5 mM EDTA, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 10 mM NaF, 10 mM disodium pyrophosphate, 5 g/ml leupeptin, 0.7 g/ml pepstatin A, 10 g/ml benzamidine). Lysed cells from two wells (800 l) were transferred to 1.5-ml tubes on ice and rotated on an inversion wheel for 1 h. Solubilized material was transferred to Beckman TLA 100.2 tubes for centrifugation at 200,000 ϫ g for 15 min at 4°C. The supernatant was transferred to 1.5-ml tubes on ice with 100 l of protein A-Sepharose beads (Amersham Biosciences) in 3% bovine serum albumin and radioimmune precipitation buffer. An aliquot of supernatant was removed for protein determination (Bio-Rad DC protein assay kit). After a 1-h pre-clearing, beads were pelleted and the supernatant was transferred to 1.5-ml tubes with 100 l of protein A-Sepharose beads and 16 g of monoclonal IgG-M2-FLAG (Kodak). Samples were placed on an inversion wheel at 4°C. After 2 h, beads were pelleted and supernatant was discarded. Beads were washed three times with ice-cold radioimmune precipitation buffer. SDS-polyacrylamide gel electrophoresis sample buffer was added to each sample to provide the same membrane protein/volume of sample for gel loading. Immune complexes were dissociated by heating to 65°C for 10 min and resolved on a 1-mm-thick, 10% SDS-polyacrylamide gel. Dried gels were analyzed quantitatively with a Molecular Dynamics PhosphorImager.
In plasmid co-transfection experiments, receptor expression was determined by flow cytometry analysis of a sample from each transfection group. The fluorescence was determined by incubation for 1 h at 37°C with monoclonal IgG-M2-FLAG (1:500 dilution, Kodak), three washes with phosphate-buffered saline, and detection with Fc-specific, fluorescein-labeled goat anti-mouse antibody (1:200 dilution, Sigma). Cells were then washed, removed from the plate with 10 mM Tris, pH 7.4, 5 mM EDTA, and fixed with 3.6% formaldehyde. Samples were analyzed within 1 h on a Becton-Dickson flow cytometer. Base-line fluorescence was determined from a sample of HEK 293 cells untransfected and/or a sample of HEK 293 cells transfected with the rat VIP receptor but not exposed to primary antibody. Base-line fluorescence was subtracted from each sample. Receptor fluorescence was normalized to total cellular protein determined from an aliquot of each transfection sample before immunoprecipitation. Gel lanes were loaded with the same amount of receptor protein.

Characterization of VPAC 1 Receptor Constructs: Receptor
Binding, Signaling, and Sequestration-Binding studies with cell membranes prepared from HEK 293 cells transiently transfected with wild-type rat VPAC 1 and the N-terminal FLAG VPAC 1 receptor cDNA resulted in an identical K D for VIP binding (4 nM, data not shown). The wild-type and Nterminal FLAG VIP type 1 receptor produced an EC 50 for cAMP accumulation of 0.2 nM in response to vasoactive intestinal polypeptide (Fig. 1A). Time-course experiments revealed a rapid increase in cAMP accumulation that declined with a t1 ⁄2 of 5 min, yielding complete cessation of further cAMP accumulation after ϳ10 -15 min of agonist exposure, for both receptors when overexpressed in HEK 293 cells (Fig. 1B). By fluorescence-activated cell sorting analysis, the epitope-tagged VPAC 1 receptor demonstrated a concomitantly rapid time course of internalization, with a t1 ⁄2 of 5 min and maximum loss of 50% by 15-20 min (Fig. 1C). Because the FLAG-tagged VIP type 1 receptor construct was similar to that of the wild-type receptor, we employed the FLAG tagged receptor to investigate VPAC 1 regulation.
Vasoactive Intestinal Polypeptide (VPAC 1 ) Receptor Phosphorylation-Receptor phosphorylation has been found to be important in the regulation of many GPCRs (6,21). Using the epitope-tagged VPAC 1 receptor, we observed a dose-dependent VPAC 1 phosphorylation in response to agonist (Fig. 2A). The major phosphorylated protein in receptor-expressing cells migrated as a broad band of 60 -80 kDa and is not present in immunoprecipitates from cells not expressing the epitopetagged VPAC 1 (Fig. 2B, far right lanes). Agonist-dependent receptor phosphorylation occurs with an EC 50 of 1.1 nM ( Fig.  2A). Interestingly, in cells not stimulated with agonist, there is a significant amount of basal receptor phosphorylation.
The Role of G Protein-coupled Receptor Kinases in VPAC 1 Receptor Desensitization and Phosphorylation-We have previously shown that the secretin receptor, another class II GPCR, exhibits GRK specificity in its desensitization. In HEK 293 cells, GRKs 2, 3, and 5 attenuate secretin receptor signaling; however, GRKs 4 and 6 do not (9). VIP receptor signaling was attenuated in HEK 293 cells overexpressing FLAG-tagged VIP type 1 receptor by GRKs 2, 3, 5, and 6, as measured by cAMP accumulation after exposure to agonist for 10 min (Fig. 3, A and  B). GRK 4 was relatively ineffective in diminishing VPAC 1 signaling (Fig. 3B). Using immunoprecipitation of the receptor, we studied the effect of overexpressed GRKs on VPAC 1 phosphorylation. In contrast to the secretin receptor, all the GRKs produced an increase in VPAC 1 receptor phosphorylation (Fig.  4, A and B). PhosphorImager analysis of immunoprecipitated VPAC 1 demonstrated that all the GRKs increased receptor phosphorylation 3-4-fold following stimulation with agonist for 10 min. The VPAC 1 receptor undergoes a variable amount of GRK-dependent basal phosphorylation that hinders comparison of GRK specificity in cells overexpressing the rat VPAC 1 receptor (Fig. 4).
The Role of G Protein-coupled Receptor Kinases 4, 5, and 6 in the Desensitization of the Endogenous VPAC 1 Receptor in HEK 293 Cells-We designed primers specifically to discriminate between the human VPAC 1 and VPAC 2 receptors. Using these primers and RT-PCR, we determined that HEK 293 cells endogenously express only the VPAC 1 receptor. We then investigated the role of GRKs in the regulation of the endogenous VPAC 1 receptor. To determine the specificity of GRK-dependent regulation of the endogenous VPAC 1 , we overexpressed GRKs 4, 5, and 6 in HEK 293 cells. As shown in Fig. 5A, overexpressing various amounts of GRK 5 or 6 caused attenuation of cAMP accumulation in response to VIP. The effects of GRKs 5 and 6 were dose-dependent, and decreasing amounts of DNA transfected corresponded to decreased levels of expressed proteins (Fig. 5B). GRK 4 did not cause a significant change in cAMP accumulation, even at very high doses of transfected DNA and expressed protein.
The Role of ␤-Arrestin and Dynamin in VPAC 1 Sequestration-␤-Arrestin binds preferentially to phosphorylated receptor and promotes receptor internalization (22). The effect of ␤-arrestin on VPAC 1 R signaling was initially studied on the endogenous VPAC 1 R in HEK 293 cells overexpressing ␤-arrestin 1 and 2. As shown in Fig. 6, ␤-arrestins 1 and 2 decreased agonist-stimulated VPAC 1 R cAMP accumulation by 27 and 39% of control levels, respectively. Neither arrestin construct caused a significant shift in EC 50 .
To further examine the role of ␤-arrestin in VPAC 1 receptor internalization, we used transiently transfected HEK 293 cells overexpressing the FLAG-tagged VPAC 1 receptor and ␤-arrestin-1, dynamin I, or their respective dominant negative mutants, ␤-arrestin-1 V53D or dynamin I K44A. Similar to results with the ␤ 2 -adrenergic receptor, overexpression of neither ␤-arrestin nor dynamin altered VPAC 1 R internalization when quantitated by fluorescence-activated cell sorting analysis as loss of cell surface receptor (Fig. 7). However, when the dominant negative constructs (␤-arrestin V53D, dynamin 1 K44A) were overexpressed, both decreased internalization of the ␤ 2adrenergic receptor, but only dynamin K44A reduced VPAC 1 receptor endocytosis (Fig. 7). Although the VPAC 1 R behaves differently from the ␤ 2 -adrenergic receptor, other receptors have shown this variance (8,23,24). This difference may be caused in part by different receptor affinities for ␤-arrestin. If the affinity of the VPAC 1 receptor for ␤-arrestin is significantly higher than that of the ␤ 2 -adrenergic receptor, then the overexpressed V53D protein may not be able to compete with endogenous ␤-arrestin and the endogenous ␤-arrestin may be able to target the receptor to clathrin-coated pits. In an attempt to resolve this, we performed additional experiments in COS 7 cells. These cells are known to have less ␤-arrestin than HEK 293 cells. Sequestration of the VPAC 1 R in these cells revealed  a similar pattern (data not shown). To investigate the role of ␤-arrestin in VPAC 1 receptor internalization, we used immunofluorescence to study the fate of the VPAC 1 receptor and ␤-arrestin after agonist stimulation.
␤-Arrestin Translocation, Vasoactive Intestinal Polypeptide Receptor Sequestration, and VPAC 1 R/␤-Arrestin Colocalization by Immunofluorescence Microscopy and Immunoprecipitation-We stimulated HEK 293 cells overexpressing the VPAC 1 receptor and observed rapid translocation of a ␤-arrestin-GFP fusion protein from the cytosol to the plasma membrane (Fig.  8A). ␤-Arrestin-GFP translocated from the cytosol to the plasma membrane robustly after stimulation with VIP, but only in cells overexpressing the VPAC 1 R. To determine more directly whether ␤-arrestin is targeted to the VPAC 1 receptor, we transfected HEK 293 cells with the epitope-tagged VPAC 1 receptor and ␤-arrestin-GFP. After 1 min of agonist exposure, the ␤-arrestin-GFP was found co-localized with the VPAC 1 receptor at the cell membrane (Fig. 8B). Similarly, after 30 min of agonist exposure, ␤-arrestin was clearly co-localized with the VPAC 1 receptor in endocytic vesicles inside the cell (Fig. 8B).
To support an interaction of the VPAC 1 receptor with ␤-arrestin, we immunoprecipitated the VIP receptor and tested for co-immunoprecipitation of overexpressed ␤-arrestin. HEK 293 cells transiently transfected with both FLAG-tagged wild-type VIP receptor and ␤-arrestin were stimulated with 100 nM VIP in a 37°C incubator for 5, 10, or 20 min. One plate of HEK 293 cells was transfected with ␤-arrestin only and served as the control. As demonstrated in Fig. 8C, immunoprecipitation of the VIP receptor brings down ␤-arrestin, suggesting the presence of a receptor-arrestin complex. DISCUSSION Although class II GPCRs are abundant and involved in the regulation of a variety of physiological processes, information on their regulation lags that known for the larger class I rhodopsin/adrenergic family of receptors. Study of GPCRs has relied on overexpressed heterologous cell systems that provide a controlled manner to investigate various aspects of receptor signal regulation. However, in some cases the overexpressed cell system may obscure molecular determinants involved in specific receptor regulation. For example, overexpression of a receptor not normally found in a specific cell type may not recapitulate receptor regulation in vivo. Similarly, receptor overexpression may alter endogenous regulatory proteins and either increase or decrease the phosphorylation of the receptor under study. Therefore, study of cell systems with endogenous receptor expression may provide the opportunity to ask specific questions not possible in the heterologous system.
Using the VPAC 1 R, we have investigated its regulation by overexpression in HEK 293 cells and by studying an endogenously expressed receptor. Placement of the FLAG epitope at the N terminus of VPAC 1 R did not significantly alter receptor binding or signaling properties, providing a useful tool in the study of VPAC 1 receptor regulation. Prior investigators have noted diminished agonist binding to the VPAC 1 with mutation of the N terminus (25, 26). We placed the epitope at the amino terminus of the processed portion of the mature receptor to minimize interactions with sites important for agonist binding. The VPAC 1 R, like all class II receptors, is coupled to Gs and activates adenylyl cyclase, and appears to follow the paradigm of regulation established for class I GPCRs (27). In this paper we demonstrate that agonist-dependent receptor phosphorylation, arrestin translocation, and consequent receptor internalization, regulates the VPAC 1 receptor. By immunoprecipitation, we demonstrate agonist-dependent VPAC 1 phosphorylation with a EC 50 of 1.1 nM. Our receptor immunoprecipitation revealed a broad band of ϳ70 kDa, similar to that seen by Fabre et al. (28).
Agonist-induced receptor phosphorylation occurs by either GRKs or second messenger-dependent kinases. The VPAC 1 receptor, when overexpressed, is rather promiscuous with respect to phosphorylation. Like many GPCRs, the VPAC 1 is phosphorylated and desensitized by GRKs 2, 3, and 5. However, when studied in an endogenously expressed fashion, GRKs 5 and 6, but not GRK 4, desensitize it preferentially. It is notable that, although GRK 4 can phosphorylate VPAC 1 receptor, when both proteins are overexpressed, GRK 4 appears incapable of functionally desensitizing the endogenously expressed VPAC 1 receptor. This is in marked contrast to the regulation of the related secretin receptor, where both GRK 4 and GRK 6 appear incapable of phosphorylating or desensitizing this receptor (9). Thus, VPAC 1 receptor is among the relatively few receptors that have been shown to be regulated by GRK 6 (27).
Phosphorylation and desensitization of the human VIP type 2 (VPAC 2 ) receptor has been demonstrated in response to agonist (29). In that study the authors demonstrated that the VPAC 2 receptor is desensitized and phosphorylated by a kinase sensitive to the cAMP-dependent protein kinase inhibitor bisindolylmaleimide (29). These authors postulated a role for GRK-dependent receptor phosphorylation; however, the role of GRKs in the phosphorylation and desensitization of the VPAC 2 receptor has not been reported. In this paper we have demonstrated that each member of the GRK family can phosphorylate HEK 293 cells overexpressing either ␤-arrestin 1 or ␤-arrestin 2 alone were exposed, in a dose-dependent manner, to VIP for 10 min to determine their ability to attenuate signaling of the endogenous VPAC 1 R. The VPAC 1 R was not overexpressed in these experiments. ␤-Arrestin 1 elicited a 27% reduction, whereas ␤-arrestin 2 reduced maximal cAMP levels by 39% of control. However, neither arrestin construct caused a significant shift in the EC 50 for cAMP accumulation. Data are mean Ϯ S.E. of three independent experiments, each done in triplicate.
FIG. 7. Effect of ␤-arrestin and dynamin on VPAC 1 R internalization. HEK 293 cells were transiently transfected with wild-type VPAC 1 and ␤-arrestin 1, ␤-arrestin 1 mutant (V53D), dynamin, or the dominant-negative dynamin mutant (K44A) on the agonist promoted sequestration of the VPAC 1 and the ␤ 2 AR as assessed by flow cytometry. FLAG-tagged VIP receptor or hemagglutinin-tagged ␤ 2 AR was transiently transfected in HEK 293 cells with 5 g of the following: empty vector (Empty), pCMV rat ␤-arrestin 1 (␤arr1), pcDNA1-AMP rat ␤-arrestin-1-V53D (V53D) (10), 8 g of pCB1 rat dynamin 1 (Dynamin), or 8 g of rat dynamin 1-K44A (K44A). Expression of mutant and wild-type ␤-arrestin-1 was monitored by immunoblot using an antibody for ␤-arrestin-1 (31). Expression of mutant and wild-type dynamin was monitored by immunoblot using an antibody for dynamin 1 (33). The data represent the mean Ϯ S.E. of at least three independent experiments, with each point done in duplicate, for each group. the VPAC 1 receptor. However, overexpression of the VPAC 1 receptor produced a significant amount of basal phosphorylation, and this may make studies on GRK-specific receptor phosphorylation less sensitive when tested in heterologous cell systems. HEK 293 cells possess an endogenous, and previously uncharacterized, VIP-type receptor. The presence of an endogenously expressing receptor provided a means to study the regulation of the VPAC 1 receptor at lower, and consistently reproducible, levels of expression. Using RT-PCR we identified this receptor to be the VPAC 1 receptor. Overexpression of GPCRs has been known to produce agonist-independent receptor phosphorylation, and this may be because of activation of mechanisms responsible for receptor regulation. Using the endogenous VPAC 1 receptor, we demonstrated GRK specificity by dose-dependent GRK expression and cAMP accumulation. The novelty of our study resides in the utility of the endogenous receptor to determine GRK specificity that would not have been evident in the classical approach using immunoprecipitation of overexpressed receptors from cells overexpressing individual GRKs. Additionally, in this case, the lack of GRK inhibitors precludes studying specificity by kinase inhibition. We had attempted to show secretin receptor GRK specificity by titrating GRK expression but were not successful. In those studies any amount of receptor overexpression resulted in an inability to produce graduated GRK expression. Furthermore, the lack of an endogenous receptor precluded the determination of receptor regulation similar to that obtained here for the VIP receptor.
Receptor phosphorylation causes ␤-arrestin translocation to many GPCRs (30), and ␤-arrestin was originally characterized as a desensitization protein (31). We investigated the role of ␤-arrestin in the regulation of the VPAC 1 by demonstrating profound and swift translocation of ␤-arrestin-GFP from the cytosol to the plasma membrane. Interestingly, when ␤-arrestin 1 or 2 were overexpressed in HEK 293 cells expressing only the endogenous receptor, either ␤-arrestin caused only a minor decrease in cAMP accumulation. This lack of effect on receptor signal termination is distinct from the effect of ␤-arrestin on GRK phosphorylated ␤ 2 -adrenergic receptors (22). This prompted us to investigate the role of ␤-arrestin in VIP recep-FIG. 8. Agonist stimulation causes a rapid translocation of ␤-arrestin GFP to the plasma membrane and the VIP receptor colocalizes with ␤-arrestin in endocytic vesicles and by immunoprecipitation. A, HEK 293 cells overexpressing the VIP receptor and a ␤-arrestin-GFP conjugate were exposed to VIP (0.1 M VIP) for the time indicated and followed with confocal microscopy. Within seconds of agonist exposure, the ␤-arrestin-GFP translocated from the cytosol (as shown at time 0) to the plasma membrane (as shown at 1 min). Translocation is rapid and persists from many minutes. B, the VIP receptor internalizes and is colocalized with ␤-arrestin in endocytic vesicles. Top panels, HEK 293 cells overexpressing the VIP receptor and ␤-arrestin-GFP prior to agonist exposure. In the left panel, the VIP receptor is localized to the plasma membrane (Texas Red, red). In the center panel, ␤-arrestin-GFP is distributed throughout the cytosol (green fluorescent protein, green). The right panel demonstrates no overlap (yellow) between VIP receptor staining and green fluorescent protein prior to agonist exposure. Middle panel, after exposure to VIP for 30 min, the VIP receptor is found within the cytosol located in vesicles (left panel). Similarly, ␤-arrestin-GFP has coalesced into vesicles within the cytoplasm (center panel) and as shown in the overlay, VIP receptor and ␤-arrestin are now co-localized in these endocytic vesicles (right panel). Lower panels, At higher magnification, the co-localization of the VIP receptor and ␤-arrestin-GFP is more evident in large doughnut shaped vesicles. C, HEK 293 cells transiently transfected with 3 g of cDNA for FLAG-tagged wild-type VIP receptor and 2 g of cDNA for ␤-arrestin were stimulated with 100 nM VIP in a 37°C incubator for 5, 10, or 20 min. One plate of HEK 293 cells was transfected only with 2 g of cDNA ␤-arrestin and served as the control (lane 1). After stimulation, cells were lysed. Supernatant was immunoprecipitated with IgG-M2-FLAG antibody and separated on a 10% SDS-PAGE gel. After separation, proteins were transferred to nitrocellulose and immunoblotted with antibody to ␤-arrestin. An aliquot of the total cellular material was assayed for ␤-arrestin, and this is shown in the bottom panel.
tor trafficking, as it has recently been proposed that ␤-arrestin serves to target GPCRs to endocytic pathways (5).
We have demonstrated a role for ␤-arrestin in the regulation of VPAC 1 receptor regulation by co-localization of the receptor with arrestin using confocal microscopy and receptor-arrestin complex formation using immunoprecipitation. Clearly, ␤-arrestin forms a complex with the VPAC 1 receptor and this complex is transported into the cell during receptor endocytosis. This internalization of the receptor-arrestin complex may play a role in subsequent receptor signaling and appears to be maintained for some time during endocytic vesicle trafficking.
The molecular determinants for GPCR internalization include ␤-arrestin and dynamin, which act to promote GPCR targeting to clathrin-coated pit-dependent endocytosis (5). However, recent studies on the secretin receptor and the angiotensin II type 1A receptor suggest that all GPCRs may not manifest the same dependence on these components of receptor internalization (8,23,24). In HEK 293 cells overexpressing the VPAC 1 receptor, agonist stimulation causes receptor internalization in a prompt manner. Receptor internalization appears to occur via endocytic vesicles. Internalization of the VPAC 1 receptor is altered by overexpression of the dynamin GTPasedeficient mutant (K44A), but not by overexpression of the wildtype dynamin protein. This supports a dynamin-dependent clathrin-coated pit path for VPAC 1 receptor internalization and supports a role for clathrin-dependent receptor trafficking that was not apparent in studies on the secretin receptor.
Another significant difference between the VPAC 1 R and the secretin receptor is the result obtained using the dominantnegative inhibitor of dynamin. Secretin receptor internalization was not effected by overexpression of K44A dynamin. Similarly, the muscarinic (M2) and angiotensin type 1A receptors internalize in the face of overexpression of this impaired dynamin protein. This finding has been used to support the hypothesis that certain GPCRs may use a dynamin-independent mechanism for receptor trafficking. However, our data on VPAC 1 R internalization support a recent observation on the muscarinic and angiotensin type 1A receptors that, under appropriate conditions, the M2 and angiotensin type 1A receptors sequester in a dynamin-dependent manner and, once activated, these GPCRs are targeted to clathrin-coated pits that are pinched off at the plasma membrane by dynamin (32).
The VPAC 1 receptor, as a member of a distinct class of GPCRs, is phosphorylated in an agonist-dependent manner by specific GRKs and internalized via clathrin-coated pits. Unlike the secretin receptor and many other GPCRs, the VPAC 1 receptor is phosphorylated and desensitized by GRK 6, information that would not have been clear by limited study in the overexpressed cell system. The utility of an endogenously expressing receptor provides a novel mechanism to pursue information on GPCR regulation. Study of endogenously expressing proteins is likely to yield results that more closely recapitulate regulation in vivo. Therefore, it appears VPAC 1 receptor regu-lation follows a paradigm similar to that of the superfamily of class I GPCRs.